Herpesviruses are a major source of morbidity and mortality in humans and their success is due to their ability to establish latent infections, and to modulate the immunity of their hosts. These abilities render the virus refractory to cure by chemotherapeutic agents, and to clearance by the immune system. While latency and immunity to herpesviruses have been intensely studied, the mechanisms that control establishment, maintenance and reactivation of latency remain obscure, especially for herpes simplex virus (HSV). This is due, in part, to the high complexities of in vivo (mostly mouse) models that while highly informative, fail to inform precisely about the unique relationship between HSV and the neuron, its target cell for latency. Recently, our understanding of latency has been significantly enhanced by the development of physiologically relevant in vitro systems employing primary cultured neurons that can be used in combination with more traditional in vivo systems to interrogate the molecular basis of latency and immunity. The over-arching approach of Project 3 is to use and develop relevant in vitro and in vivo models systems to demonstrate roles for intrinsic, innate, and adaptive immunity and viral countermeasures that determine the balance of the latency/reactivation axis. Aim 1, with Projects 1 and 2, examines the structure and function of autophagosomes clusters (intrinsic immunity) that are induced in sensory neurons during the establishment and maintenance of latency. We will probe these newly-discovered structures with high resolution microscopy, and characterize gene expression and chromatin patterns of HSV in cluster-positive neurons. Aim 2 examines the role of viral modulation of reactive oxygen species and mitochondrial movement in cultured neurons on the ability of HSV to establish, and reactivate from latency, and on the genesis of the interferon-dependent innate immune response. With Projects 1 and 2, this aim will also examine the roles of HSV proteins ICP0 and ICP34.5 in the modulation of these responses and their effects on gene expression and chromatin patterns. Aim 3 addresses the hypothesis that antibodies are key immune determinants of HSV-1 replication, pathogenesis, and latency in the nervous system. The activities of antibodies within sensory ganglia will be defined, and their roles in the control of replication, latency, and reactivation, including effects on gene expression and chromatin, will be determined. With Project 1, the ability of an experimental vaccine and antibodies to protect mice not only from mortality following HSV challenge, but also from the long-lived behavioral pathology associated with neonatal infection will be assessed. The general approach then is to continue collaborations with Projects 1 and 2, and, using Core B, to develop and exploit our primary neuron and neonatal infection model systems interfaced with analyses with Core A of gene expression and chromatin patterns, and experimental immunization. These approaches will determine how different branches of immunity control latency, and how HSV counters these responses. This framework will identify novel pathways to help intervene in the lifecycle of this persistent human pathogen.